Photonic neural probe enabled microendoscopes for light-sheet light-field computational fluorescence brain imaging

Abstract. Significance Light-sheet fluorescence microscopy is widely used for high-speed, high-contrast, volumetric imaging. Application of this technique to in vivo brain imaging in non-transparent organisms has been limited by the geometric constraints of conventional light-sheet microscopes, which require orthogonal fluorescence excitation and collection objectives. We have recently demonstrated implantable photonic neural probes that emit addressable light sheets at depth in brain tissue, miniaturizing the excitation optics. Here, we propose a microendoscope consisting of a light-sheet neural probe packaged together with miniaturized fluorescence collection optics based on an image fiber bundle for lensless, light-field, computational fluorescence imaging. Aim Foundry-fabricated, silicon-based, light-sheet neural probes can be packaged together with commercially available image fiber bundles to form microendoscopes for light-sheet light-field fluorescence imaging at depth in brain tissue. Approach Prototype microendoscopes were developed using light-sheet neural probes with five addressable sheets and image fiber bundles. Fluorescence imaging with the microendoscopes was tested with fluorescent beads suspended in agarose and fixed mouse brain tissue. Results Volumetric light-sheet light-field fluorescence imaging was demonstrated using the microendoscopes. Increased imaging depth and enhanced reconstruction accuracy were observed relative to epi-illumination light-field imaging using only a fiber bundle. Conclusions Our work offers a solution toward volumetric fluorescence imaging of brain tissue with a compact size and high contrast. The proof-of-concept demonstrations herein illustrate the operating principles and methods of the imaging approach, providing a foundation for future investigations of photonic neural probe enabled microendoscopes for deep-brain fluorescence imaging in vivo.

Photograph showing attachment of an image fiber bundle to a photonic neural probe and the corresponding apparatus.The neural probe was fixed onto a probe holder/carrier using 5 min epoxy and the probe holder was mounted under a microscope.(b) Side-view photograph of the next step in the process where the fiber bundle was aligned over top of the neural probe using a translation stage.(c) Top-down optical micrograph of the microendoscope tip following the next step where the fiber bundle was epoxied to the neural probe.The neural probe tip is visible in (b) where the fiber bundle is placed parallel to and on top of the shanks, while ensuring the bundle facet is in close proximity to the grating coupler (GC) emitters, and specifically, in close proximity to the most proximal row of GCs (Sheet 1).The attachment was encapsulated by UV epoxy.A multicore fiber with 16 cores is shown here as an example, and 10-core fiber was used in the microendoscopes.(c) Top-down optical micrographs of the neural probe chip facet (left) and shanks (right) with the multicore fiber aligned to the array of fiber-to-chip waveguide edge couplers on the neural probe.When selected by the scanning system, each core of the multicore fiber couples laser light to an edge coupler of the neural probe (left) and the light is routed to a row of GCs on the shanks where it is emitted as a light sheet (right).As noted in Sec.2.3 of the manuscript, a neural probe with a "half-sheet" design (with each sheet spanning 2 adjacent shanks) was used here.One of the shanks broke during packaging, affecting half of the sheets.The images in the left column were not affected, and the sample was illuminated by a sheet spanning 2 shanks.However, the images in the right column were affected, and the sample was illuminated by a sheet generated by 1 shank.

Note 1 .
Fig S1 Microendoscope fiber bundle attachment.(a)Photograph showing attachment of an image fiber bundle to a photonic neural probe and the corresponding apparatus.The neural probe was fixed onto a probe holder/carrier using 5 min epoxy and the probe holder was mounted under a microscope.(b) Side-view photograph of the next step in the process where the fiber bundle was aligned over top of the neural probe using a translation stage.(c) Top-down optical micrograph of the microendoscope tip following the next step where the fiber bundle was epoxied to the neural probe.The neural probe tip is visible in (b) where the fiber bundle is placed parallel to and on top of the shanks, while ensuring the bundle facet is in close proximity to the grating coupler (GC) emitters, and specifically, in close proximity to the most proximal row of GCs (Sheet 1).The attachment was encapsulated by UV epoxy.

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Fig S2 Visible-light multicore fiber alignment.(a) Top-down photograph of the optical scanning system used to address the cores of the multicore fiber.(b) Optical micrographs of the facet of a multicore fiber coupled to the scanning system; light is coupled into different cores of the fiber via actuation of the MEMS mirror (spatial addressing).A multicore fiber with 16 cores is shown here as an example, and 10-core fiber was used in the microendoscopes.(c) Top-down optical micrographs of the neural probe chip facet (left) and shanks (right) with the multicore fiber aligned to the array of fiber-to-chip waveguide edge couplers on the neural probe.When selected by the scanning system, each core of the multicore fiber couples laser light to an edge coupler of the neural probe (left) and the light is routed to a row of GCs on the shanks where it is emitted as a light sheet (right).

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Fig S3 Multicore fiber attachment.(a) Side-view photograph during application of UV epoxy to attach the multicore fiber to the neural probe chip facet.The multicore fiber was fixed in a ferrule for stability of the alignment and strength of the final package.(b) Top-and side-view photographs after completion of the packaging process.(c) Photograph of the microendoscope imaging system.Laser light is coupled into the neural probe through the multicore fiber and the proximal end of the fiber bundle is imaged with an epifluorescence microscope.An image of the fully-packaged microendoscope captured by a USB camera is shown on the background screen.A micromanipulator was used to control the microendoscopic insertion into samples.
Fig S4 Light sheet characterization in a fluorescein solution.(a) and (b) Photographs showing the distal end of the packaged LSLF microendoscope immersed in a fluorescein solution.(c) Side-view photograph of the resultant fluorescence from a light sheet emitted by the microendoscope into the fluorescein solution.(d) Fluorescence images of the light sheet profiles of the microendoscope.The dashed red lines delineate the top surface of the shanks.Scale bars: 200 µm.The sheet thicknesses in these side profile measurements are overestimated due to the collection of outof-focus light by the microscope (i.e., the sheet width was larger than the microscope depth of focus).The secondary upward-pointing beams observed in (b), (c), and Sheets 3 to 5 in (d) were due to second-order diffraction from the GC emitters as discussed in Sec.4.3 of the manuscript.These beams were only visible at high laser powers and their optical powers were approximately 7×, 5×, and 4× lower than the those of the primary light sheets for Sheets 3 to 5, respectively.Microendoscope 1 was used for the light sheet characterization in fluorescein.

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Fig S5 Free-space light-sheet profile measurements.(a) Illustration of the light sheet profile measurement approach (not to scale).A coverslip coated with a fluorescent thin film on one side was positioned above and parallel to the neural probe shanks.A light sheet emitted by the neural probe was incident on the thin film, and the resultant fluorescence, corresponding to a cross-section of the light sheet, was imaged by a widefield microscope above the coverslip.The distance between the fluorescent thin film and the neural probe was varied to image the light sheet cross-section at various propagation distances.(b) Light sheet cross-sections imaged at discrete light sheet propagation distances, L prop .All images have been intensity normalized for visibility.Scale bars: 100 µm.The optical emissions from the row of GCs corresponding to the light sheet merged with increasing propagation distance, synthesizing a semiuniform sheet with averaged FWHM thickness < 11 µm, < 17 µm, < 20 µm for L prop of 100 µm, 300 µm, and 500 µm, respectively.Microendoscope 1 was used for the light sheet characterization in free space.

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Fig S10 Microendoscope LSLF images of fluorescent beads suspended in agarose.Images corresponding to Sheets 1 to 4 are shown in the first to fourth rows, respectively.The first column shows the raw images after fiber bundle core interpolation; the second and third columns present reconstructed images at two arbitrary depth planes of the focal stack with ≈30 µm axial (z) spacing.The x, y, and z-directions are indicated.All images have been intensity-adjusted for visibility.Scale bars: 50 µm.Microendoscope 1 was used for these imaging tests.